Journal: The FASEB Journal

Article Title: Nuclear respiratory factor 1 (NRF‐1) upregulates the expression and function of reduced folate carrier (RFC) at the blood‐brain barrier

doi: 10.1096/fj.202000239rr

Figure Lengend Snippet: FIGURE 6 Involvement of NRF-1 in the transcriptional regulation of human SLC19A1. A, Predicted positions of NRF-1 binding sites in the human SLC19A1 promoter were determined using the JASPAR database.28 B, ChIP analysis was performed using normal rabbit IgG (NRI-IP) or rabbit polyclonal anti-NRF-1 antibody (NRF1-IP) from hCMEC/D3 cells treated with 5 μM of PQQ for 24 hours. PQQ-treated cells showed significantly elevated NRF-1 occupancy to all three predicted NRF-1 binding sites (Sites 1-3) compared to NRI-IP control. As a negative control, a nonspecific region within the SLC19A1 promoter 20 kb upstream from the transcription start site (TSS) was amplified. No significant difference in NRF-1 occupancy was observed at the nonspecific region relative to NRI-IP control. Results are shown as a percentage of input DNA from n = 5 independent experiments (*P < .05, **P < .01, ***P < .001)

Article Snippet: Specific human or mouse primer pairs for: SLC19A1/Slc19a1 (RFC; Hs00953344_m1, Mm00446220_m1), NRF1/Nrf1 (NRF1; Hs00602161_m1, Mm01135606_m1), PPARGC1A/ Ppargc1a (PGC-1α; Hs00173304_m1, Mm01208835_m1), Tfam (Tfam; Hs01082775_m1, Mm00447485_m1), TFB1M/ Tfb1m (TFB1M; Hs01084404_m1, Mm00524825_m1), TFB2M/Tfb2m (TFB2M; Hs00915025_m1, Mm01620397_ s1), ABCB1 (P-gp; Hs00184500_m1), ABCG2 (BCRP; Hs01053790_m1), ABCC2 (MRP2; Hs00166123_m1), and ABCC3 (MRP3; Hs00978473_m1), were designed and validated by Life Technologies for use with TaqMan qPCR chemistry.

Techniques: Binding Assay, Control, Negative Control, Amplification

Journal: bioRxiv

Article Title: Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

doi: 10.1101/2020.03.16.993600

Figure Lengend Snippet: a . Domain organization of human FAM111A. b . Immunoblot analysis of stable U2OS cell lines treated with Doxycycline (DOX) to induce expression of WT or mutant forms of GFP-FAM111A. c . DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n >2000 cells per condition). d . Cells treated as in (c) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC ( n >2000 cells per condition). e - g . Quantification of data in (d) (red bars, mean). Cells in S phase were identified based on EdU positivity. h . Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s 0 =1). i . U2OS or U2OS/ ∆FAMIIIA cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies. j . U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies. k . As in (d), except that cells were stained with RFC1 antibody ( n >2000 cells per condition). l . Quantification of data in (k) for S phase (EdU-positive) cells (red bars, mean). Data are representative of at least three ( b - g , i - l ) and two ( h ) independent experiments with similar outcomes.

Article Snippet: Antibodies used for immunofluorescence included: γH2AX (05-636 (Clone JBW301), Millipore (1:500)), FAM111A (ab184572, Abcam (1:300)), MCM2 (610701, Clone 46/BM28, BD Transduction Lab (1:500)), PCNA (#2037, Triolab Immunoconcepts (1:500)), RFC1 (sc-271656, Santa Cruz (1:300) or ab3566, Abcam (1:1,000)), RPA2 (NA19L (Clone Ab-3), Roche (1:1,000)), α-Tubulin (T9026, Sigma-Aldrich (1:5,000)).

Techniques: Western Blot, Expressing, Mutagenesis, Labeling, Staining, Cytometry, Immunoprecipitation, Mass Spectrometry, Control, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

doi: 10.1101/2020.03.16.993600

Figure Lengend Snippet: a . Overview of heterozygous FAM111A mutations found in patients with gracile bone dysplasia or Kenny-Caffey syndrome. b . Immunoblot analysis of U2OS cell lines treated with DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT(low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in and . c . U2OS/GFP-FAM111A cell lines treated with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. d . Quantification of data in (c) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n >2000 cells per condition). e . As in (c), except that cells were pulse-labeled with EU. f . Quantification of EU incorporation in cells in (e) (red bars, mean; n >2000 cells per condition). g . DOX-treated U2OS/FAM111A cell lines were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC. h . As in (g), except that cells were stained with RPB1 antibody. i . Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated with DOX in the absence or presence of Z-VAD-FMK. j . As in (i), but using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles. k . In vitro auto-cleavage assay. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h and analyzed by immunoblotting. l . GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s 0 =1). Data are representative of at least three ( b - k ) and two ( l ) independent experiments with similar outcomes.

Article Snippet: Antibodies used for immunofluorescence included: γH2AX (05-636 (Clone JBW301), Millipore (1:500)), FAM111A (ab184572, Abcam (1:300)), MCM2 (610701, Clone 46/BM28, BD Transduction Lab (1:500)), PCNA (#2037, Triolab Immunoconcepts (1:500)), RFC1 (sc-271656, Santa Cruz (1:300) or ab3566, Abcam (1:1,000)), RPA2 (NA19L (Clone Ab-3), Roche (1:1,000)), α-Tubulin (T9026, Sigma-Aldrich (1:5,000)).

Techniques: Western Blot, Expressing, Labeling, Staining, In Vitro, Cleavage Assay, Purification, Recombinant, Incubation, Mutagenesis, Mass Spectrometry

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: A Immunoblot analysis of stable U2OS cell lines left untreated or incubated with DOX to induce expression of WT or mutant forms of GFP‐FAM111A. B DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse‐labeled with EdU, and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image‐based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6A . C Cells treated as in (B) were pre‐extracted, fixed, and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC ( n > 2,000 cells per condition). D–F Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Fig S6B–D . G Analysis of FAM111A interactors. U2OS/GFP‐FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP), and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/−DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). H U2OS or U2OS/ ΔFAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies. I U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies. J As in (C), except that cells were stained with RFC1 antibody ( n > 2,000 cells per condition). K Quantification of data in (J) for S phase (EdU‐positive) cells (red bars, mean). Data information: Data are representative of at least three (A–F, H–K) and two (G) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Mutagenesis, Labeling, Staining, Cytometry, Immunoprecipitation, Mass Spectrometry, Control, Transfection, Plasmid Preparation

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Validation of FAM111A interactors identified by mass spectrometry (Fig G; ). U2OS/GFP‐FAM111A cell lines treated or not with DOX were subjected to GFP immunoprecipitation (IP) followed by immunoblotting with indicated antibodies. U2OS cells transfected with constructs expressing indicated FLAG‐tagged RFC1 fragments were subjected to FLAG IP followed by immunoblotting with indicated antibodies. U2OS cells transfected with non‐targeting control (CTRL) or FAM111A siRNA were pre‐extracted, fixed, and stained with FAM111A antibody. U2OS cells labeled with EdU were pre‐extracted, fixed, and stained with indicated antibodies. Endogenous FAM111A localizes to nucleoli in G1 phase and G2 phase (EdU‐negative) cells and relocates to nuclear foci in S phase (EdU‐positive) cells. U2OS cells transfected with indicated siRNAs were pre‐extracted, fixed, and stained with FAM111A and RFC1 antibodies. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX. Scale bars, 10 μm. Data information: Data (A–F) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Biomarker Discovery, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Control, Staining, Labeling

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Overview of heterozygous FAM111A mutations found in patients with gracile bone dysplasia or Kenny–Caffey syndrome. Immunoblot analysis of U2OS cell lines left untreated or incubated with DOX to induce expression of the indicated GFP‐FAM111A alleles. U2OS/GFP‐FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP‐FAM111A WT cells used in Figs and (see Fig A). U2OS/GFP‐FAM111A cell lines treated or not with DOX, pulse‐labeled with EdU, and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. Quantification of data in (C) for S phase (EdU‐positive) cells (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6J . As in (C), except that cells were pulse‐labeled with EU. Quantification of EU incorporation in cells in (E) (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6L . U2OS/GFP‐FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC. As in (G), except that cells were stained with RPB1 antibody. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX in the absence or presence of Z‐VAD-FMK. As in (I), using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles. Purified recombinant FLAG‐FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto‐proteolytic activity was analyzed by immunoblotting. GFP IPs from U2OS cell lines expressing GFP‐FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). Data information: Data are representative of at least three (B–K) and two (L) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Labeling, Staining, Purification, Recombinant, Activity Assay, Mutagenesis, Mass Spectrometry

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines conditionally expressing GFP‐FAM111A WT at different levels. Cells in (A) were treated with DOX for 16 h, pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition; A.U., arbitrary units). As in (B), except that cells were treated with DOX for 24 h. Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines expressing WT or patient‐associated GFP‐FAM111A alleles. Cells in (D) were pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Quantification of data in (E) (red bars, mean). Representative images of U2OS/GFP‐FAM111A cell lines that were treated or not with DOX for the indicated times, fixed, and co‐stained with PCNA and RPA2 antibodies. Scale bar, 10 μm. Quantification of data in (G) (gray bars, average; n > 2,000 cells per condition). U2OS/GFP‐FAM111A cell lines treated or not with DOX were stained with γ‐H2AX antibody and analyzed for γ‐H2AX signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6K . As in (I), except that cells were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 signal intensity in S phase cells (gated based on DAPI signal intensity) was analyzed by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6M . As in (I), except that cells were stained with RPB1 antibody and analyzed for RPB1 signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). U2OS cell lines conditionally expressing untagged ectopic FAM111A alleles were treated or not with DOX for 24 h, labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Immunoblot analysis of stable U2OS/FAM111A cell lines transfected or not with FAM111A siRNA targeting the 3′UTR, and subsequently treated or not with DOX to express ectopic untagged FAM111A alleles. Data information: Data (A–M) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Stable Transfection, Expressing, Labeling, Staining, Transfection

Journal: Nature

Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

doi: 10.1038/s41586-019-1553-0

Figure Lengend Snippet: SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.

Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

Techniques: Expressing, Transduction, Control, Flow Cytometry, Plasmid Preparation, Incubation, Comparison, Two Tailed Test

Journal: Nature

Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

doi: 10.1038/s41586-019-1553-0

Figure Lengend Snippet: a , Thin layer chromatography (TLC) analysis of [ 32 P] ATP standard (STD) and enzymatically synthesized [ 32 P] 2’3’-cGAMP. 2’3’-cGAMP was purified on STING resin. Unbound nucleotides flowed through the resin (STING FT). Following four washes, the bound [32P] 2’3’-cGAMP was eluted over three fractions. b , DRaCALA binding analysis of [ 32 P] 2’3’-cGAMP to STING C-terminal domain (CTD) in the presence of competing unlabeled nucleotides (200 μM). c , Thin layer chromatography (TLC) analysis of [ 32 P]-ATP and enzymatically synthesized [ 32 P]-2’3’-cGAMP and [ 32 P]-c-di-AMP. d , Binding titration of [ 32 P] 2’3’-cGAMP or [ 32 P] c-di-AMP to mSTING C-Terminal Domain (CTD), determined with DRaCALA assays. Red dashed lines represent the 95% confidence interval for the non-linear regression. e , Time course of [ 32 P] 2’3’-cGAMP (left panel) or [ 32 P] 3’3’-CDA (right panel) uptake in THP-1 monocytes. f , TLC analysis (left panel) and STING-binding (DRaCALA) (right panel) of [ 32 P] ATP standard, or [ 32 P] 2’3’-cGAMP recovered from supernatants of THP-1 monocytes at the indicated time points. g , h , Effect of cell culture medium pH on [ 32 P] 2’3’-cGAMP uptake in THP-1 (g) monocytes or U937 monocytes (h). i , Time course of [ 32 P] 2’3’-cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 expression vector. j, mRNA expression levels of SLC19A1 (SLC.) in K562 cells expressing control shRNAs (sh1 and sh2) or an SLC19A1 -targeting shRNA (sh9). k, mRNA expression levels of CXCL10 in K562 cells described in panel j, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. l, [ 3 H]-Methotrexate uptake in K562 cells described in panel j, 1h after exposure to [ 3 H]-Methotrexate. m , Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells described in panel j. n , Time course of [ 32 P] 2’3’-cGAMP uptake in U937 monocytes in the presence or absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. o . Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells in the presence or absence of 500 μM competing, unlabeled (anti-) folates or sulfasalazine. p , Competition uptake assay of [ 32 P] 2’3’-cGAMP uptake in THP-1 cells in the presence of varying concentrations of competing, unlabeled 5-me-THF (IC50 = 4.10 ± 0.16 nM), methotrexate (IC50 = 54.83 ± 5.08 nM), 2’3’-cGAMP (IC50 = 1.89 ± 0.11 μM), sulfasalazine ((IC50 = 2.06 ± 0.17 μM), and folic acid (IC50 = 4.79 ± 0.08 μM). q , Trans-stimulation of [ 32 P] 2’3’-cGAMP influx in THP-1 cells by 5-me-THF. Cells were preloaded with indicated concentrations of 5-me-THF for 30 min. Cells were washed and incubated with [ 32 P] 2’3’-cGAMP for one hour. r , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated human PBMCs from four healthy donors. In panel a and c, data are representative of three independent experiments with similar results. In panel b, data are means of n=2 technical replicates and are representative of three independent experiments. In panel d and f, data are means of n=2 technical replicates and are representative of two independent experiments. In panel e and m, data are means ± SD of n=3 technical replicates and are representative of three independent experiments. In panel g, h, i, and n-r, data are means ± n=3 technical replicates and are representative of two independent experiments. In panel j and k, data are means ± SEM of n=3 biologically independent experiments. In panel l, data are means of n=2 biologically independent experiments.

Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

Techniques: Thin Layer Chromatography, Synthesized, Purification, Binding Assay, Titration, Cell Culture, Plasmid Preparation, Expressing, Control, shRNA, Incubation

Journal: Nature

Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

doi: 10.1038/s41586-019-1553-0

Figure Lengend Snippet: a, mRNA expression levels of Slc19a1 in mouse L1210 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh4 and sh6). b, mRNA expression levels of Cxcl10 in L1210 cells described in panel a stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. c, [ 3 H]-Methotrexate uptake in L1210 cells described in panel a 1h after exposure to [ 3 H]-Methotrexate. d, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells described in panel a. e, mRNA expression levels of Slc19a1 in mouse C1498 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh6). f, mRNA expression levels of Cxcl10 in the C1498 cells described in panel e, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. g, [ 3 H]-Methotrexate uptake in C1498 cells described in panel e 1h after exposure to [ 3 H]-Methotrexate. h , Time course of [ 32 P] 2’3’-cGAMP uptake in C1498 cells transduced with a non-targeting control shRNA (control) or Slc19a1 shRNA. i, j, mRNA expression levels of Slc19a1 in mouse bone marrow-derived macrophages (BMM) (i) or mouse bone marrow-derived dendritic cells (BMDCs) (j) transduced or not with control shRNAs (sh1 and 2) or an shRNA targeting Slc19a1 . k, l, mRNA expression of the Cxcl10 in cells described in panels i and j stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. m, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. n, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes pretreated or not with NHS-methotrexate (MTX) (5 μM). o, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells pretreated or not with NHS-MTX (5 μM). In panels a-c, e-g, j and l, data are means of n=2 biologically independent experiments. In panel d, h, i, k, n and o, data are means ± SD of n=3 technical replicates and are representative of two independent experiments. In panel m, data are means of n=2 technical replicates and are representative of two independent experiments. In time course experiments (d, h, m, n, o), data are presented as counts per minute (cpm) normalized to cell count.

Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

Techniques: Expressing, Control, shRNA, Transduction, Derivative Assay, Cell Counting

Journal: Nature

Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

doi: 10.1038/s41586-019-1553-0

Figure Lengend Snippet: SLC19A1 transports CDNs. a , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by K562 cells transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control shRNA (control) or SLC19A1 shRNA (right panel). c , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated THP-1 (left panel) and K562 (right panel) cells. d , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes in the presence and absence of 100 μM competing, unlabeled cyclic dinucleotides (left panel) or 200 μM competing, unlabeled nucleotides (right panel). e , Time course of [ 32 P] 2’3’-cGAMP uptake in THP-1 monocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. f , Normalized [ 32 P] 2’3’-cGAMP uptake after three hours in human PBMCs from six healthy donors in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. g , Coomassie staining and western blot analysis of pulldowns by 2’3’-cGAMP (+) or control (−) Sepharose of mSTING-CTD or hSLC19A1. h , Western blot analysis of hSLC19A1 affinity purification (AP) with 2’3’-cGAMP Sepharose in the absence (−) or presence (+) of free, unbound 2’3’-cGAMP, 5-me-THF, or methotrexate (250 μM). In panels a-d, data are means ± SEM of n=3 biological replicates. In panel e, data are means ± SD of n=3 technical replicates and data are representative of three independent experiments. In panel f, data are means ± SD of n=6 healthy donors conducted over two independent experiments. Panels g and h are representative of two independent experiments; for gel source data, see . Statistical analyses were performed using unpaired, two-tailed Student’s t-tests (a-d), or a one-way ANOVA followed by a Tukey’s post-test (f). **** P ≤ 0.0001.

Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

Techniques: Transduction, Plasmid Preparation, Control, Expressing, shRNA, Staining, Western Blot, Affinity Purification, Two Tailed Test

Journal: Nature

Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

doi: 10.1038/s41586-019-1553-0

Figure Lengend Snippet: a , Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie blue staining of His-tagged human SLC19A1 (Ni-NTA affinity-purified) pull-downs with Sepharose beads coupled with 2’3’-cGAMP (+) or control Sepharose beads (−). Input is shown in the right panel. b , Western blots of the samples in a with anti-SLC19A1 antibody. c , Pull-downs of SLC19A1 competed with CDNs. His-tagged SLC19A1 was incubated with no CDN or with the indicated competing CDNs (250 μM) before pulldowns with 2’3’-cGAMP-Sepharose, followed by SDS-PAGE and Western blotting with an anti-SLC19A1 antibody. A pulldown with control Sepharose is shown for comparison. For gel source data, see . d , SDS-PAGE analysis followed by Coomassie blue staining of pull-downs of mSTING-C-Terminal Domain (CTD) with 2’3’-cGAMP (+) or control (−) Sepharose. In all panels, data are representative of two independent experiments with similar results.

Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

Techniques: SDS Page, Staining, Affinity Purification, Control, Western Blot, Incubation, Comparison

Journal: Molecular pharmaceutics

Article Title: Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier

doi: 10.1021/acs.molpharmaceut.7b00572

Figure Lengend Snippet: Relative expression of major folate transport systems in various in vitro and ex vivo models of the BBB. (A) mRNA expression of human, rat, or mouse SLC19A1/Slc19a1 (RFC), SLC46A1/Slc46a1 (PCFT), and FOLR1/Folr1 (FRα) genes were determined in immortalized (hCMEC/D3) and primary (hBMEC) cultures of human brain microvessel endothelial cells, immortalized cultures of rat brain microvessel endothelial cells (RBE4), and rodent brain capillaries using TaqMan gene expression assay. Results are presented as mean relative mRNA expression ± SEM normalized to the housekeeping human/rat/mouse cyclophilin B gene from n = 3 independent experiments. (B) Immunoblot analysis of RFC and PCFT protein expression was performed in the same BBB model systems. HEK293 and HeLa cells served as positive controls, while actin was used as a loading control. Multiple protein bands for RFC and PCFT are indicative of differential glycosylation of these transmembrane proteins. A representative blot is shown from n = 3 independent experiments.

Article Snippet: Specific human/rat/mouse primer pairs for SLC19A1 / Slc19a1 (RFC; Hs00953344_m1, Rn00446220_m1, Mm00446220_m1), SLC46A1 / Slc46a1 (PCFT; Hs00560565_m1, Rn01471182_m1, Mm00546630_m1), FOLR1 / Folr1 (FR α ; Hs01124179_91, Rn00591759_m1, Mm00433355_m1), and NRI1I / Nri1i (VDR; Hs01045843_m1, Mm00437297_m1) were designed and validated by Life Technologies for use with TaqMan qPCR chemistry.

Techniques: Expressing, In Vitro, Ex Vivo, Gene Expression, Western Blot, Control, Glycoproteomics

Journal: Molecular pharmaceutics

Article Title: Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier

doi: 10.1021/acs.molpharmaceut.7b00572

Figure Lengend Snippet: Effect of calcitriol treatment on RFC expression in hCMEC/D3 cells. (A) mRNA expression of human NRI1I/rodent Nri1i gene was determined in immortalized cultures of human brain microvessel endothelial (hCMEC/D3) cells and isolated mouse brain capillaries using TaqMan gene expression assay. Results are presented as mean relative mRNA expression ± SEM normalized to the housekeeping human cyclophilin B gene from n = 3 independent experiments. Significant increases in SLC19A1 mRNA (B) and RFC protein (C) expression were observed in hCMEC/D3 cells treated with calcitriol (50–500 nM) for 6 or 24 h compared to vehicle (ethanol) control. Multiple protein bands for RFC (63–75 kDa) are indicative of differential glycosylation of the transmembrane protein. Rat kidney lysates served as positive control, while actin was used as a loading control. Results are presented as mean ± SEM for n = 3–4 independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: Specific human/rat/mouse primer pairs for SLC19A1 / Slc19a1 (RFC; Hs00953344_m1, Rn00446220_m1, Mm00446220_m1), SLC46A1 / Slc46a1 (PCFT; Hs00560565_m1, Rn01471182_m1, Mm00546630_m1), FOLR1 / Folr1 (FR α ; Hs01124179_91, Rn00591759_m1, Mm00433355_m1), and NRI1I / Nri1i (VDR; Hs01045843_m1, Mm00437297_m1) were designed and validated by Life Technologies for use with TaqMan qPCR chemistry.

Techniques: Expressing, Isolation, Gene Expression, Control, Glycoproteomics, Positive Control

Journal: Molecular pharmaceutics

Article Title: Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier

doi: 10.1021/acs.molpharmaceut.7b00572

Figure Lengend Snippet: Effect of calcitriol treatment on RFC expression in isolated mouse brain capillaries. Significant increases in SLC19A1 mRNA (A) and RFC protein (B) expression were observed in mouse brain capillaries treated with 100 nM calcitriol for 4 h compared to vehicle (ethanol) control. Results are presented as mean ± SEM for n = 3 independent experiments, where each experiment contained pooled brain tissues from 3 to 4 animals per group. *, p < 0.05.

Article Snippet: Specific human/rat/mouse primer pairs for SLC19A1 / Slc19a1 (RFC; Hs00953344_m1, Rn00446220_m1, Mm00446220_m1), SLC46A1 / Slc46a1 (PCFT; Hs00560565_m1, Rn01471182_m1, Mm00546630_m1), FOLR1 / Folr1 (FR α ; Hs01124179_91, Rn00591759_m1, Mm00433355_m1), and NRI1I / Nri1i (VDR; Hs01045843_m1, Mm00437297_m1) were designed and validated by Life Technologies for use with TaqMan qPCR chemistry.

Techniques: Expressing, Isolation, Control